Regional transcriptomic profiling reveals immune system enrichment in nonfailing atria and all chambers of the failing human heart.

The different chambers of the human heart demonstrate regional physiological traits and may be differentially affected during pathological remodeling, resulting in heart failure. Few previous studies, however, have characterized the different chambers at a transcriptomic level. We, therefore, conducted whole tissue RNA sequencing and gene set enrichment analysis of biopsies collected from the four chambers of adult failing (n = 8) and nonfailing (n = 11) human hearts. Atria and ventricles demonstrated distinct transcriptional patterns. When compared with nonfailing ventricles, the transcriptional pattern of nonfailing atria was enriched for many gene sets associated with cardiogenesis, the immune system and bone morphogenetic protein (BMP), transforming growth factor-ß (TGF-ß), MAPK/JNK, and Wnt signaling. Differences between failing and nonfailing hearts were also determined. The transcriptional pattern of failing atria was distinct compared with that of nonfailing atria and enriched for gene sets associated with the innate and adaptive immune system, TGF-ß/SMAD signaling, and changes in endothelial, smooth muscle cell, and cardiomyocyte physiology. Failing ventricles were also enriched for gene sets associated with the immune system. Based on the transcriptomic patterns, upstream regulators associated with heart failure were identified. These included many immune response factors predicted to be similarly activated for all chambers of failing hearts. In summary, the heart chambers demonstrate distinct transcriptional patterns that differ between failing and nonfailing hearts. Immune system signaling may be a hallmark of all four heart chambers in failing hearts and could constitute a novel therapeutic target.NEW & NOTEWORTHY The transcriptomic patterns of the four heart chambers were characterized in failing and nonfailing human hearts. Both nonfailing atria had distinct transcriptomic patterns characterized by cardiogenesis, the immune system and BMP/TGF-ß, MAPK/JNK, and Wnt signaling. Failing atria and ventricles were enriched for gene sets associated with the innate and adaptive immune system. Key upstream regulators associated with heart failure were identified, including activated immune response elements, which may constitute novel therapeutic targets.

Prevention of cardiovascular disease in young adults: Focus on gender differences. A collaborative review from the EAS Young Fellows.

A steady rise in cardiovascular morbidity and mortality has been observed in young adults within the last decades. This trend corresponds to an increasing prevalence of traditional cardiovascular risk factors such as obesity and diabetes mellitus type 2 among young adults living in developed countries. Moreover, age-specific risk factors, such as substance abuse, contraceptive medication, and pregnancy-related diseases also correlate with an increased incidence of cardiovascular diseases. In this review, we discuss the available data for young adults on the epidemiology and the rationale for the causality of traditional and newly emerging risk factors of atherosclerotic cardiovascular diseases. We focus on gender-related differences in the exposure to these risk factors, investigate the recent data regarding screening and risk stratification in the young adult population, and describe the current state of the art on lifestyle and therapeutic intervention strategies in the primary prevention setting.

Metagenomic sequencing of human cardiac tissue reveals Microbial RNA which correlates with Toll-like receptor-associated inflammation in patients with heart disease.

Cardiovascular disease (CVD) is strongly associated with chronic low-grade inflammation, involving activated Toll-like receptors and their downstream cellular machinery. Moreover, CVD and other related inflammatory conditions are associated with infiltration of bacteria and viruses originating from distant body sites. Thus, in this study we aimed to map the presence of microbes in the myocardium of patients with heart disease that we previously found to display upregulated Toll-like receptor signaling. We performed metagenomics analysis of atrial cardiac tissue from patients undergoing coronary artery bypass grafting (CABG) or aortic valve replacement (AVR) and compared with atrial cardiac tissue from organ donors. A total of 119 species of bacteria and seven species of virus were detected in the cardiac tissue. RNA expression of five bacterial species were increased in the patient group of which L. kefiranofaciens correlated positively with cardiac Toll-like receptor-associated inflammation. Interaction network analysis revealed four main gene set clusters involving cell growth and proliferation, Notch signaling, G protein signaling and cell communication in association with L. kefiranofaciens RNA expression. Taken together, intracardial expression of L. kefiranofaciens RNA correlates with pro-inflammatory markers in the diseased cardiac atrium and may have an effect on specific signaling processes important for cell growth, proliferation and cell communication.

Subtyping of cardiac amyloidosis by mass spectrometry-based proteomics of endomyocardial biopsies.

BACKGROUND: Cardiac amyloidosis is a severe condition leading to restrictive cardiomyopathy and heart failure. Mass spectrometry-based methods for cardiac amyloid subtyping have become important diagnostic tools but are currently used only in a few reference laboratories. Such methods include laser-capture microdissection to ensure the specific analysis of amyloid deposits. Here we introduce a direct proteomics-based method for subtyping of cardiac amyloidosis. METHODS: Endomyocardial biopsies were retrospectively analysed from fresh frozen material of 78 patients with cardiac amyloidosis and from 12 biopsies of unused donor heart explants. Cryostat sections were digested with trypsin and analysed with liquid chromatography - mass spectrometry, and data were evaluated by proteomic software. RESULTS: With a diagnostic threshold set to 70% for each of the four most common amyloid proteins affecting the heart (LC κ, LC λ, TTR and SAA), 65 of the cases (87%) could be diagnosed, and of these, 61 cases (94%) were in concordance with the original diagnoses. The specimens were also analysed for the summed intensities of the amyloid signature proteins (ApoE, ApoA-IV and SAP). The intensities were significantly higher (p < 0.001) for all assigned cases compared with controls. CONCLUSION: Cardiac amyloidosis can be successfully subtyped without the prior enrichment of amyloid deposits with laser microdissection.

Human intracardiac SSEA4+CD34 cells show features of cycling, immature cardiomyocytes and are distinct from Side Population and C-kit+CD45- cells.

Cardiomyocyte proliferation has emerged as the main source of new cardiomyocytes in the adult. Progenitor cell populations may on the other hand contribute to the renewal of other cell types, including endothelial and smooth muscle cells. The phenotypes of immature cell populations in the adult human heart have not been extensively explored. We therefore investigated whether SSEA4+CD34- cells might constitute immature cycling cardiomyocytes in the adult failing and non-failing human heart. The phenotypes of Side Population (SP) and C-kit+CD45- progenitor cells were also analyzed. Biopsies from the four heart chambers were obtained from patients with end-stage heart failure as well as organ donors without chronic heart failure. Freshly dissociated cells underwent flow cytometric analysis and sorting. SSEA4+CD34- cells expressed high levels of cardiomyocyte, stem cell and proliferation markers. This pattern resembles that of cycling, immature, cardiomyocytes, which may be important in endogenous cardiac regeneration. SSEA4+CD34- cells isolated from failing hearts tended to express lower levels of cardiomyocyte markers as well as higher levels of stem cell markers. C-kit+CD45- and SP CD45- cells expressed high levels of endothelial and stem cell markers-corresponding to endothelial progenitor cells involved in endothelial renewal.

Overexpression of the SARS-CoV-2 receptor angiotensin converting enzyme 2 in cardiomyocytes of failing hearts.

Hospitalized patients who die from Covid-19 often have pre-existing heart disease. The SARS-CoV-2 virus is dependent on the ACE2 receptor to be able to infect cells. It is possible that the strong link between cardiovascular comorbidities and a poor outcome following a SARS-CoV-2 infection is sometimes due to viral myocarditis. The aim was to examine the expression of ACE2 in normal hearts and hearts from patients with terminal heart failure. The ACE2 expression was measured by global quantitative proteomics and RT-qPCR in left ventricular (LV) tissue from explanted hearts. Immunohistochemistry was used to examine ACE2 expression in cardiomyocytes, fibroblasts and endothelial cells. In total, tissue from 14 organ donors and 11 patients with terminal heart failure were included. ACE2 expression was 2.6 times higher in 4 hearts from patients with terminal heart failure compared with 6 healthy donor hearts. The results were confirmed by immunohistochemistry where more than half of cardiomyocytes or fibroblasts showed expression of ACE2 in hearts from patients with terminal heart failure. In healthy donor hearts ACE2 was not expressed or found in few fibroblasts. A small subpopulation of endothelial cells expressed ACE2 in both groups. Upregulated ACE2 expression in cardiomyocytes may increase the risk of SARS-CoV-2 myocarditis in patients with heart failure.

Markedly reduced myocardial expression of γ-protocadherins and long non-coding RNAs in patients with heart disease.

BACKGROUND: Adverse cardiac remodeling and tissue damage following heart disease is strongly associated with chronic low grade inflammation. The mechanisms underlying persisting inflammatory signals are not fully understood, but may involve defective and/or non-responsive transcriptional and post-transcriptional regulatory mechanisms. In the current study, we aimed to identify novel mediators and pathways involved in processes associated with inflammation in the development and maintenance of cardiac disease. METHODS AND RESULTS: We performed RNA sequencing analysis of cardiac tissue from patients undergoing coronary artery bypass grafting (CABG) or aortic valve replacement (AVR) and compared with control tissue from multi-organ donors. Our results confirmed previous findings of a marked upregulated inflammatory state, but more importantly, we found pronounced reduction of non-protein coding genes, particularly long non-coding RNAs (lncRNA), including several lncRNAs known to be associated with inflammation and/or cardiovascular disease. In addition, Gene Set Enrichment Analysis revealed markedly downregulated microRNA pathways, resulting in aberrant expression of other genes, particularly γ-protocadherins. CONCLUSIONS: Our data suggest that aberrant expression of non-coding gene regulators comprise crucial keys in the progression of heart disease, and may be pivotal for chronic low grade inflammation associated with cardiac dysfunction. By unmasking atypical γ-protocadherin expression as a prospective genetic biomarker of myocardial dysfunction, our study provides new insight into the complex molecular framework of heart disease. Creating new approaches to modify non-coding gene regulators, such as those identified in the current study, may define novel strategies to shift γ-protocadherin expression, thereby normalizing part of the molecular architecture associated with heart disease.

COMP (Cartilage Oligomeric Matrix Protein) Neoepitope: A Novel Biomarker to Identify Symptomatic Carotid Stenosis.

OBJECTIVE: COMP (cartilage oligomeric matrix protein) is abundantly expressed in the cardiovascular system, cartilage, and atherosclerotic plaques. We investigated if the total COMP (COMPtotal) and COMP neoepitope (COMPneo) with other cardiovascular markers and clinical parameters could identify symptomatic carotid stenosis. Approach and Results: Blood samples were collected from patients with symptomatic carotid stenosis (stenosis, n=50), patients with stroke without carotid stenosis but small plaques (plaque, n=50), and control subjects (n=50). COMPtotal and COMPneo were measured using an ELISA. Ninety-two cardiovascular disease markers were measured by the Olink CVD kit. The presence of native COMP and COMPneo was determined by immunohistochemistry. The concentration of COMPneo was higher and COMPtotal was lower in the stenosis group. When the concentration was compared between the stenosis and control groups, IL-1ra (interleukin-1 receptor antagonist protein), IL6 (interleukin-6), REN (Renin), MMP1 (matrix metalloproteinase-1), TRAIL-R2 (tumor necrosis factor-related apoptosis-inducing ligand receptor 2), ITGB1BP2 (integrin beta 1 binding protein 2), and COMPneo were predictive of stenosis. Conversely, KLK6 (kallikrein-6), COMPtotal, NEMO (nuclear factor-kappa-B essential modulator), SRC (Proto-oncogene tyrosine-protein kinase Src), SIRT2 (SIR2-like protein), CD40 (cluster of differentiation 40), TF (tissue factor), MP (myoglobin), and RAGE (receptor for advanced glycation end-products) were predictive of the control group. Model reproducibility was good with the receiver operating characteristic plot area under the curve being 0.86. When comparing the plaque group and stenosis group, COMPneo, GAL (galanin), and PTX3 (pentraxin-related protein PTX3) were predictive of stenosis. Model reproducibility was excellent (receiver operating characteristic plot area under the curve 0.92). COMPneo was detected in smooth muscle-, endothelial-, and foam-cells in carotid stenosis. CONCLUSIONS: Degradation of COMP may be associated with atherosclerosis progression and generation of a specific COMP fragment-COMPneo. This may represent a novel biomarker that together with COMPtotal and other risk-markers could be used to identify symptomatic carotid stenosis. Graphic Abstract: A graphic abstract is available for this article.

Wide QRS-T angles are associated with markers of increased inflammatory activity independently of hypertension and diabetes.

BACKGROUND: Wide QRS-T angles and inflammatory activity are markers of future cardiovascular events including sudden cardiac death (SCD). The association between wide QRS-T angles and inflammatory activation is however not fully understood. METHODS: 1,094 study participants of both sexes, 50-64 years old, were included from a randomly selected population-based cohort as a part of the Swedish CArdioPulmonary bioImage Study (SCAPIS) pilot study. Serum samples were analyzed for markers of inflammation, cardiac wall stress/injury, and the metabolic syndrome. Wide QRS-T angles were defined using Frank vectorcardiography. Variables were analyzed through unsupervised principal component analysis (PCA) as well as Orthogonal Projections to Latent Structures (OPLS) modeling. In addition, a subset of study participants was analyzed in a post hoc matched group design. RESULTS: Wide QRS-T angles correlated positively with markers of inflammation, cardiac wall stress/injury, the metabolic syndrome, and male sex in both PCA and OPLS models. In the matched post hoc analysis, participants with wide QRS-T angles had significantly higher counts of white blood cells (WBC) and neutrophils in comparison with matched controls. WBC as well as the number of neutrophils, monocytes, basophils, eosinophils and levels of C-reactive protein, IL-1, IL-4, IL-6, TNF-α, and NT-pro-BNP were also significantly higher in comparison with healthy controls. CONCLUSIONS: Markers of inflammatory activation and cardiac injury/wall stress were significantly higher in the presence of wide QRS-T angles. These results corroborate an association between abnormal electrophysiological function and inflammatory activation and may have implications for the prediction of SCD.

A Possible Mechanism behind Faster Clearance and Higher Peak Concentrations of Cardiac Troponin I Compared with Troponin T in Acute Myocardial Infarction.

BACKGROUND: Although cardiac troponin I (cTnI) and troponin T (cTnT) form a complex in the human myocardium and bind to thin filaments in the sarcomere, cTnI often reaches higher concentrations and returns to normal concentrations faster than cTnT in patients with acute myocardial infarction (MI). METHODS: We compared the overall clearance of cTnT and cTnI in rats and in patients with heart failure and examined the release of cTnT and cTnI from damaged human cardiac tissue in vitro. RESULTS: Ground rat heart tissue was injected into the quadriceps muscle in rats to simulate myocardial damage with a defined onset. cTnT and cTnI peaked at the same time after injection. cTnI returned to baseline concentrations after 54 h, compared with 168 h for cTnT. There was no difference in the rate of clearance of solubilized cTnT or cTnI after intravenous or intramuscular injection. Renal clearance of cTnT and cTnI was similar in 7 heart failure patients. cTnI was degraded and released faster and reached higher concentrations than cTnT when human cardiac tissue was incubated in 37°C plasma. CONCLUSION: Once cTnI and cTnT are released to the circulation, there seems to be no difference in clearance. However, cTnI is degraded and released faster than cTnT from necrotic cardiac tissue. Faster degradation and release may be the main reason why cTnI reaches higher peak concentrations and returns to normal concentrations faster in patients with MI.

Glycogenin is Dispensable for Glycogen Synthesis in Human Muscle, and Glycogenin Deficiency Causes Polyglucosan Storage.

CONTEXT: Glycogenin is considered to be an essential primer for glycogen biosynthesis. Nevertheless, patients with glycogenin-1 deficiency due to biallelic GYG1 (NM_004130.3) mutations can store glycogen in muscle. Glycogenin-2 has been suggested as an alternative primer for glycogen synthesis in patients with glycogenin-1 deficiency. OBJECTIVE: The objective of this article is to investigate the importance of glycogenin-1 and glycogenin-2 for glycogen synthesis in skeletal and cardiac muscle. DESIGN, SETTING, AND PATIENTS: Glycogenin-1 and glycogenin-2 expression was analyzed by Western blot, mass spectrometry, and immunohistochemistry in liver, heart, and skeletal muscle from controls and in skeletal and cardiac muscle from patients with glycogenin-1 deficiency. RESULTS: Glycogenin-1 and glycogenin-2 both were found to be expressed in the liver, but only glycogenin-1 was identified in heart and skeletal muscle from controls. In patients with truncating GYG1 mutations, neither glycogenin-1 nor glycogenin-2 was expressed in skeletal muscle. However, nonfunctional glycogenin-1 but not glycogenin-2 was identified in cardiac muscle from patients with cardiomyopathy due to GYG1 missense mutations. By immunohistochemistry, the mutated glycogenin-1 colocalized with the storage of glycogen and polyglucosan in cardiomyocytes. CONCLUSIONS: Glycogen can be synthesized in the absence of glycogenin, and glycogenin-1 deficiency is not compensated for by upregulation of functional glycogenin-2. Absence of glycogenin-1 leads to the focal accumulation of glycogen and polyglucosan in skeletal muscle fibers. Expression of mutated glycogenin-1 in the heart is deleterious, and it leads to storage of abnormal glycogen and cardiomyopathy.

Individuals with familial hypercholesterolemia and cardiovascular events have higher circulating Lp(a) levels.

BACKGROUND: Cardiovascular disease (CVD) is a major cause of mortality and morbidity. Increased low-density lipoprotein cholesterol (LDL-C) level is its major risk factor. Familial hypercholesterolemia (FH) is a genetic disorder characterized by elevated LDL-C since birth and subsequent premature CVD. There is a heterogeneity in the CVD onset in patients with FH. This is potentially due to the presence of other independent risk factors. Lipoprotein(a) [Lp(a)] is an LDL-like particle and represents a strong risk factor for CVD. OBJECTIVE: Our objective was to understand the contribution of Lp(a) in the susceptibility to CVD in individuals with genetic diagnosis of FH. METHODS: We measured Lp(a) levels in 2 independent and well-characterized genetic-FH cohorts: the FH-Gothenburg cohort (n = 190) and the FH-CEGP Milan cohort (n = 160). The genetic diagnosis was performed by targeted next-generation sequencing (FH-Gothenburg and part of the FH-CEGP Milan cohort), or by Sanger sequencing. RESULTS: We show that among individuals with genetic diagnosis of FH, those with previous CVD had higher Lp(a) levels. In addition, analyzing the response to the lipid-lowering therapies, we have also shown that statins had the same LDL-C-lowering effect irrespective of the type of FH-causative mutation. However, when we examined the lipid-lowering effect of proprotein convertase subtilisin/kexin type 9 inhibition by antibodies, we observed a trend in a better reduction of the LDL-C level in carriers of nonsense mutations. CONCLUSION: In conclusion, our results suggest that Lp(a) contributes to CVD onset in individuals with genetic diagnosis of FH. Our finding supports the importance to identify an efficacious therapy to lower Lp(a) in patients with FH to prevent CVD onset or recurrence.

Toll-like receptor-mediated inflammation markers are strongly induced in heart tissue in patients with cardiac disease under both ischemic and non-ischemic conditions.

BACKGROUND: A sustained low grade inflammatory state is a recognized feature of various diseases, including cardiovascular disease. This state of chronic inflammation involves activation of Toll-like receptor (TLR) signaling. However, little is known regarding the genetic profile of TLR components in cardiac tissue from patients with cardiac disease. METHODS: In this study we investigated the genetic profile of 84 TLR markers in a unique set of cardiac tissue from patients that had undergone either coronary artery bypass grafting (CABG) or aortic valve replacement (AVR). In addition, we compared the gene data from the cardiac tissue with the same gene profile in blood as well as circulating cytokines to elucidate possible targets in blood that could be used to estimate the inflammatory state of the heart in cardiac disease. RESULTS: We found a marked upregulation of TLR-induced inflammation in cardiac tissue from both patient groups compared to healthy controls. The inflammation appeared to be primarily mediated through TLR1, 3, 7, 8 and 10, resulting in a marked induction of mediators of the innate immune response. Furthermore, the gene expression data in combination with unbiased multivariate analysis suggested a difference in inflammatory response in ischemic cardiac tissue compared to non-ischemic cardiac tissue. Serum levels of IL-13 were significantly elevated in both CABG and AVR patients compared to controls, whereas other cytokines did not appear to coincide with cardiac TLR-induced inflammation. CONCLUSIONS: We propose that cardiac disease in humans may be mediated by local cardiac TLR signaling under both ischemic and non-ischemic conditions.

The Atrioventricular Junction: A Potential Niche Region for Progenitor Cells in the Adult Human Heart.

A stem cell niche is a microenvironment where stem cells reside in a quiescent state, until activated. In a previous rat model, we combined 5-bromo-2-deoxy-uridine labeling with activation of endogenous stem cells by physical exercise and revealed a distinct region, in the atrioventricular junction (AVj), with features of a stem cell niche. In this study, we aim to investigate whether a similar niche exists in the human heart. Paired biopsies from AVj and left ventricle (LV) were collected both from explanted hearts of organ donors, not used for transplantation (N = 7) and from severely failing hearts from patients undergoing heart transplantation (N = 7). Using antibodies, we investigated the expression of stem cell, hypoxia, proliferation and migration biomarkers. In the collagen-dense region of the AVj in donor hearts, progenitor markers, MDR1, SSEA4, ISL1, WT1, and hypoxia marker, HIF1-α, were clearly detected. The expression gradually decreased with distance from the valve. At the myocardium border in the AVj costaining of the proliferation marker Ki67 with cardiomyocyte nuclei marker PCM1 and cardiac Troponin-T (cTnT) indicated proliferation of small cardiomyocytes. In the same site we also detected ISL1+/WT1+/cTnT cells. In addition, heterogeneity in cardiomyocyte sizes was noted. Altogether, these findings indicate different developmental stages of cardiomyocytes below the region dense in stem cell marker expression. In patients suffering from heart failure the AVj region showed signs of impairment generally displaying much weaker or no expression of progenitor markers. We describe an anatomic structure in the human hearts, with features of a progenitor niche that coincided with the same region previously identified in rats with densely packed cells expressing progenitor and hypoxia markers. The data provided in this study indicate that the adult heart contains progenitor cells and that AVj might be a specific niche region from which the progenitors migrate at the time of regeneration.

Human cardiac fibroblasts isolated from patients with severe heart failure are immune-competent cells mediating an inflammatory response.

This study was aimed to elucidate the immunoregulatory properties of human cardiac fibroblasts cultured under pro-inflammatory and hypoxic conditions. Human heart tissue for isolating cardiac cells is generally hard to obtain, particularly from all four chambers of the same heart. Since different parts of the heart have different functions and therefore may have different immunoregulatory properties, ability to analyse cells from all chambers allows for a unique and comprehensive investigation. Cells were isolated from all four chambers of the heart from patients undergoing cardiac transplantation surgery due to severe chronic heart failure (CHF) (n = 6). Cells isolated from one donor heart, were used for comparison with the experimental group. Primary cultured human cardiac fibroblasts were treated with Lipopolysaccharide (LPS) to induce an inflammatory response. Cells were also subjected to hypoxia. To determine immunoregulatory properties of the cells, cytokine and chemokine profiles were determined using multiplex ELISA. RESULTS: On average, the fibroblasts population constituted approximately 90% of the expanded non-myocytes. Levels of cytokines and chemokines were markedly increased in human cardiac fibroblasts cultured under inflammatory conditions, with a similar response in fibroblasts from all compartments of the heart. Unexpectedly, hypoxia did not further augment cytokine and chemokine secretion. In conclusion, human cardiac fibroblasts are a robust source of pro-inflammatory mediators in the failing heart, independent of hypoxia, and might play a critical role in inflammation associated with the pathogenesis of CHF.

Hypoxic cardiac fibroblasts from failing human hearts decrease cardiomyocyte beating frequency in an ALOX15 dependent manner.

A common denominator for patients with heart failure is the correlation between elevated serum levels of proinflammatory cytokines and adverse clinical outcomes. Furthermore, lipoxygenase-induced inflammation is reportedly involved in the pathology of heart failure. Cardiac fibroblasts, which are abundant in cardiac tissue, are known to be activated by inflammation. We previously showed high expression of the lipoxygenase arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), in ischemic cardiac tissue. The exact roles of ALOX15 and 15-HETE in the pathogenesis of heart failure are however unknown. Biopsies were collected from all chambers of explanted failing human hearts from heart transplantation patients, as well as from the left ventricles from organ donors not suffering from chronic heart failure. Biopsies from the left ventricles underwent quantitative immunohistochemical analysis for ALOX15/B. Gene expression of ALOX enzymes, as well as 15-HETE levels, were examined in cardiac fibroblasts which had been cultured in either hypoxic or normoxic conditions after isolation from failing hearts. After the addition of fibroblast supernatants to human induced pluripotent stem cell-derived cardiomyocytes, intracellular calcium concentrations were measured to examine the effect of paracrine signaling on cardiomyocyte beating frequency. While ALOX15 and ALOX15B were expressed throughout failing hearts as well as in hearts from organ donors, ALOX15 was expressed at significantly higher levels in donor hearts. Hypoxia resulted in a significant increase in gene and protein expression of ALOX15 and ALOX15B in fibroblasts isolated from the different chambers of failing hearts. Finally, preconditioned medium from hypoxic fibroblasts decreased the beating frequency of human cardiomyocytes derived from induced pluripotent stem cells in an ALOX15-dependent manner. In summary, our results demonstrate that ALOX15/B signaling by hypoxic cardiac fibroblasts may play an important role in ischemic cardiomyopathy, by decreasing cardiomyocyte beating frequency.

The Arachidonate 15-Lipoxygenase Enzyme Product 15-HETE Is Present in Heart Tissue from Patients with Ischemic Heart Disease and Enhances Clot Formation.

Ischemic heart disease is a major cause of death and morbidity and the search for novel therapeutic targets is still required. We have previously shown that the enzyme arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), is highly expressed in ischemic heart tissue, but its role in the pathogenesis of ischemic heart disease is unclear. Here we showed that expression of ALOX15, but not ALOX12 or ALOX15B, was increased in ischemic versus non-ischemic human heart biopsy samples. A similar ALOX expression pattern was found in hypoxic human cardiomyocytes and cardiac endothelial cells. We also showed that levels of 15-HETE were significantly higher in ischemic versus non-ischemic human heart biopsy samples and showed a tendency to increase in serum from the patients with ischemic heart disease. Moreover, hypoxia increased the production of 15-HETE levels from human cardiomyocytes and cardiac endothelial cells. The hypoxia-induced increase in 15-HETE levels from human cardiomyocytes was inhibited by the ALOX15 inhibitor baicalein. Finally, by using intrinsic rotational thromboelastometry, we showed that human whole blood clotted faster in the presence of 15-HETE. In summary, we propose that increased ALOX15 expression in heart tissue under ischemic conditions may lead to increased production of 15-HETE, potentially contributing to thrombosis.

Intracellular flow cytometry may be combined with good quality and high sensitivity RT-qPCR analysis.

Flow cytometry (FCM) has become a well-established method for analysis of both intracellular and cell-surface proteins, while quantitative RT-PCR (RT-qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT-qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT-qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL-1. A549 cells were identified by the cell surface marker ABCG2, while HL-1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT-qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT-qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT-qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL-1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT-qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT-qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT-qPCR data of not fixed samples with those of fixed and permeabilized samples.

Physical exercise affects slow cycling cells in the rat heart and reveals a new potential niche area in the atrioventricular junction.

Physical exercise has several beneficial effects on the heart. In other tissues it has been shown to activate endogenous stem cells. There is however a lack of knowledge how exercise affects the distribution of progenitor cells as well as overall cell turnover within the heart. Therefore, proliferating cells were identified using BrdU DNA labeling in a rat exercise model. Slow cycling cells were identified by label retention. BrdU+ nuclei were counted in apex, ventricle and atrioventricular junction (AV junction), as well as in skin tissue where label retaining cells (LRC) have been described previously. After 13 weeks of chasing, the cells with the highest intensity were identified and considered as LRC. Heart tissue showed slower proliferation compared to skin. The highest number of BrdU+ cells was found in the AV junction. Here, a sub region in close proximity to the valvular insertion point was observed, where density of BrdU+ cells was high at all time points. Physical exercise increased proliferation in AV junction at the early stage. Furthermore, the sub region was found to harbor a significant higher number of LRC compared to other regions of the heart in the exercised animals. Progenitor markers MDR1 and Sca-1 were detected in the same area by immunohistochemistry. In conclusions, our data shows that physical exercise affects cell turnover and distribution of LRC in the heart. Furthermore, it reveals a region within the AV junction of the heart that shows features of a stem cell niche.

SSEA-4+ CD34- cells in the adult human heart show the molecular characteristics of a novel cardiomyocyte progenitor population.

Stage-specific embryonic antigen (SSEA) expression is used to describe the differentiation state of an embryonic stem cell (ESC). In human ESCs, SSEA-3 and SSEA-4 are highly expressed in undifferentiated cells and downregulated upon differentiation. SSEA-4 has also been described as a marker for adult stem cells in various tissues, including human neonatal cardiac tissue. However, there is currently little data on the expression of SSEAs in human adult cardiac tissue. We obtained right and left atrial biopsies from patients undergoing cardiac surgery. These were dissociated, stained for SSEAs and other cardiac stem cell markers and analyzed by flow cytometry. Directly isolated cells expressed variable levels of SSEA-1, SSEA-3 and SSEA-4. The SSEA-1+ population was established as contaminating hematopoietic cells. The SSEA-4+ population, on the other hand, could be subdivided based on the endothelial progenitor marker CD34. The SSEA-4+ CD34- population in the right atrium had a high gene expression of both early (TBX5, NKX2.5) and late (TNNT2) cardiomyocyte markers. The SSEA-4+ CD34+ population, on the other hand, overlapped with previously described C-kit+ CD45- cardiac stem cells. Primary monolayer-cultured cells retained expression of SSEAs while the cardiomyogenic specification in the SSEA-4+ CD34- population was lost. In tissue sections, SSEA-4+ cells could be identified both within and outside the myocardium. Within the myocardium, some SSEA-4+ cells coexpressed cardiomyogenic markers. In conclusion, the results show that the adult human heart expresses SSEAs and that there is a subpopulation of SSEA-4+ CD34- cells that show features of a cardiomyocyte progenitor population.